The present invention relates to novel antibodies to the Streptococcus mutans bacteria that are naturally found in the mouth, and play a role in the development of dental caries. The invention relates to methods of detection of S. mutans using the antibodies of the invention or fragments or derivatives thereof. The invention also relates to diagnosing, monitoring, treating and protecting the teeth from dental caries using the antibodies of the invention or fragments or derivatives thereof.
Throughout this application, various publications are referenced within parentheses and cited at the end of the application. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
Currently human dental caries, or cavities, are detected by changes in translucency, color, hardness or X-ray density of teeth. These technologies have limitations both in specificity and reproducibility. Further, they do not show, at a single time point, whether or not the disease is active.
The bacterium Streptococcus mutans, or S. mutans (named and described by Clark in 1924) is known to be a prime etiologic agent for the initiation and progression of human dental caries. (Fitzgerald and Keyes, 1960; Loesche, 1982; Loesche, 1986; Tanzer, 1997). S. mutans is one of the primary factors in acid dissolution of the apatite (mineral) component of the enamel then the dentin, or of the cementum then the dentin. A strong correlation between the proportion of S. mutans in dental plaque or in saliva relative to other bacterial species and the presence or risk of future outbreaks of dental caries has been documented, (Tanzer, 1997; Krasse, 1988). Therefore, S. mutans in plaque or saliva can serve as an index for both caries activity state and caries risk or susceptibility (Loesche et al., 1975; Ellen, 1976; Krasse, 1985; Krasse, 1988). These indices play an increasingly important role in the diagnosis and treatment of dental caries (Hume, 1993; Mundorff et al., 1993; Van Houte, 1993).
Present techniques of detection and quantitative determination of S. mutans include bacterial culture with selective media using either broth or agar plate systems (Ellen, 1976; Loesche, 1982) and polymerase chain reaction techniques (Igarashi et al., 1996). Each of these methods requires significant time (on the order of days), well trained personnel and sophisticated equipment to perform. Consequently, existing techniques are relatively expensive and time consuming.
Alternatively, monoclonal antibody based detection methods allow a rapid and accurate, yet economic quantitative measurement of the presence of bacterial cells, and have significant advantages compared to traditional culture sensitivity assays or polymerase chain reaction (PCR) techniques. Bacteria produce unique polysaccharide structures of lipooligosaccharide or lipopolysaccharide or other polysaccharides, among a large range of other chemical components and products, on their cell surfaces. Monoclonal antibodies can be raised against these chemical structures, using standard hybridoma techniques (Kohler and Milstein, 1975). The sensitivity and accuracy of this method is largely dependent on the specificity of the monoclonal antibody produced. By making and screening large numbers of hybrid cell lines, one can find certain monoclonal antibodies which are species-specific for the desired bacteria, i.e. the monoclonal antibodies ICL 11 and ICL12 recognize the 0139 antigen of Vibrio cholerae (Hasan, 1994)). These monoclonal antibodies can be linked to various detection systems including, for example, fluorescent reagents, calorimetric reagents or coagglutination reagents. The resulting labeled antibodies can specifically bind to the desired bacterium in any sample, and rapidly present the result through the linked detection systems (Harlow and Lane, 1988).
TABLE 1 monoclonal PCR Culture Sensitivity antibody Reporting 2 days 7-10 days 3-10 min time send out samples send out samples instant results at chairside Cost .about.$100 .about.$100 less than $10 Precision Species level genus level Species level Test few hundred cells only cultivable cells few hundred cells sensitivity regardless of need viable cells regardless of viability viability Accuracy &gt;90% .about.50% &gt;90% Lab yes yes no requirement
Due to overwhelming advantages, monoclonal antibody-based detection methods have been widely used in medical microbiology. At present, there are nearly one hundred different monoclonal antibody based detection methods available to diagnose various pathogenic bacteria. However, to date there have been no such methods available for the detection of dental caries. While other investigators have made monoclonal antibodies to S. mutans, these monoclonal antibodies are not suited for diagnostic or clinical uses because they are not species specific for S. mutans and cannot detect the presence of S. mutans at very low levels. Almost all previous monoclonal antibodies were made against surface proteins of S. mutans (i.e. glucosyltransferase, agglutinin, surface antigen PI, etc.). Similar proteins can also be found on other Streptoccocci species. Thus, these monoclonal antibodies can not be used to distinguish S. mutans from other Streptoccocci species. To diagnose dental caries it is important to be able to discern the S. mutans strain from other bacteria that may be present. One needs to find a monoclonal antibody that is specific for a unique epitope specific to the S. mutans cell surface.